A useful protein may be generally divided into proteins for pharmaceuticals and related research such as immunomodulators, enzyme inhibitors, and hormones, or industrial proteins such as proteins for diagnosis and adding enzymes for biotransformation. A process for mass-producing these proteins necessarily requires gene expression, culture, and purification technologies. In particular, the expression technology occupies the most important part in the process for protein production, and is a key indicator for judging economic feasibility, and accordingly, the technology is a main process for increasing value-added or utility. In the related art, a method of screening bacterial host or animal cell line having high productivity, producing a desired protein from the cell line, and separating and purifying the desired protein has been used to obtain a specific protein. However, this method has problems in that an obtained amount is limited, and at the time of being applied to other proteins, the same productivity may not be guaranteed, and it is difficult to obtain products with homogenous qualities (the same protein in view of a structure and function). Recently, in accordance with development of recombinant DNA (genetic engineering) technology, a method in which a large amount of desired protein is easily obtained by transforming the useful gene into E. coli, plant cell line, or animal cell line, has been commonly used. In particular, since productivity is linked with cost, mass-production of recombinant protein using microorganisms (single cells such as E. coli, yeast, actinomyces) having an advantage of being rapidly cultured with high concentration in relatively cheap medium is known to have a significantly great advantage in view of commercial aspect. In addition, these hosts are well-defined in view of physiological/genetic characteristics, and effective methods and techniques for recombinant gene manipulation are well developed. Accordingly, due to an advantage in which desired useful protein is obtainable with high efficiency, medicinal proteins including insulin and industrial enzyme, and the like, have been produced successfully. It is known that animal/plant hosts having relatively complex physiological/genetic traits and regulatory mechanism require a difficult genetic manipulation/regulation process, such that utilization frequency thereof is relatively low in expressing a foreign gene besides a pharmaceutical protein with the purpose of application to a living body. However, at the time of universally utilizing expression regulatory mechanism, the above-described disadvantage is possible to be overcome, such that a lot of researches into discovery and improvement of an expression control method capable of being applicable equally to all eukaryotic cell lines including also prokaryotic cell lines have been conducted.
Expression systems for recombinant protein production have attempt engineering access in vectors, hosts, and ORF level, and prototypes of related and developed technique are relatively well known (Marino, M. H., Biopharm. 2 (1989) 18-33; Goeddel, D. V. et al., Methods Enzymol. 185 (1990) 3-7; Wurm, F. and Bernard, A., Curr. Opin. Biotechnol. 10 (1999) 156-159). Successful expression of a number of foreign genes has been reported by various strategies and technical modifications based on these prototype techniques; however, due to lack of generalization logic, a process of re-optimization for a portion or all of the components of an expression system depending on specific genes, has been generally required. In this process, a strategy of inducing an increase in an amount of transcript by combination of a strong promoter and regulatory elements, and a secondary structure of the transcript, and an expression strategy focusing on an increase of thermodynamic stability of the transcript are used. However, it is known that this strategy lacks generalization with the applicability to all proteins, and more fundamentally, there remains a problem in that cases where there is no significant correlation between the transcript and an amount of translated protein frequently occur. Recently, as a solution to this problem, a new strategy related with an increase in efficiency of protein translation or translational rate control is known as a potential logic that is possible to achieve generalization.
Low expression efficiency occurring in the translation step is caused by coding sequences (codon) of foreign genes having codon usage frequency showing different patterns from protein coding sequences of an innate gene which is well expressed in host cells. It is known that since codon preference in tRNA pool of a host coevoluted by making a pair of codon in a genome coding region is significantly biased, each amino acid is translated with biased codon for each cell. In addition, it is well known that bias in the process of using the codon is capable of changing peptide elongation rate (Sørensen M. A. et al., 1993). Accordingly, the most effective production for recombinant proteins needs to change and control foreign gene codon in consideration of codon usage of a host, as well as improvement at a transcription module level in consideration of vector sequence (promoter and operator) and factors interacting the vector sequence that are considered to increase transcription efficiency. As one of the various methods for solving the protein translation inhibition problem, a codon optimization technique was developed. As an attempt to increase the translation speed of a protein encoding region which is inefficient in the translation, the codon optimization technique generally uses a strategy for replacing codons (rare codons) that are rarely used or infrequently used in host cells with preferred codons.
Codon optimization is to increase translation speed by decreasing rare codon ratio in a wild type foreign gene. According to the codon optimization, an expression amount of recombinant protein is increased, which is easily achieved using a computer on the basis of a host-specific codon usage table. When the sequence of a target gene is input and the codon table of the host used in the expression is input, substitution with the codon showing a relative high frequency is made to obtain results in order of score. However, since DNA in which wild-type ORF base sequences of DNA are changed into other sequences encoding the same type of amino acids (synonymous codon) needs to be artificially synthesized, cost may be increased. In addition, various kinds of genes need to be synthesized and tested according to a codon sorting/classification method, and cases in which an improved amount of expression is not exhibited as compared to the wild-type gene, unlike expectations, are frequently observed. More extremely, it has been reported that an expression amount is more increased in codon de-optimization (strategy of changing preferred codons to rare codons by applying a logic opposite to optimization) rather than codon optimization. A method of supplementing an amount of tRNA for corresponding codon (rare codon) has also been attempted as circuitous solution of recombinant protein expression efficiency due to a difference in codon usage (Lee, Su Jeong et al., 2006). In this process, a method of additionally adding a plasmid into a host cell, the plasmid encoding specific tRNA that is insufficient in host cells has been attempted. However, this method has also a disadvantage in that it is required to provide a vector including a target gene together with the plasmid encoding the specific tRNA. In addition, intracellular stability and difficulty in obtaining a reproducible result that are fundamental problems of dual vector-expression system still remain as a challenge to be solved.
The present invention provides a universal protein over-expression tag including a ramp function as a novel method capable of increasing translation efficiency. When the ramp tag is used to increase translation efficiency, it is not necessary to change original ORF sequence, which is unlike codon optimization, such that there is no change in protein intrinsic activity, and DNA artificial synthesis according to codon substitution is not required, which is also economical in view of cost. In addition, the tag is capable of securing functional fusion with other sequences as much as possible, and is capable of guaranteeing grafting supremacy to be grafted with other functional tags. Accordingly, a technology of designing a tag capable of rapidly increasing translation efficiency at low cost has an important economic/industrial effect. In particular, it is expected that this universal expression theory (use only tag) will remarkably increase production yield of foreign protein in animal/plant cell lines that has relatively complicated regulatory mechanisms and thus has some limitations for general use.